High-Throughput Discovery and Characterization of Human Transcriptional Effectors.
Thousands of proteins localize to the nucleus; however, it remains unclear which contain transcriptional effectors. Here, we develop HT-recruit, a pooled assay where protein libraries are recruited to a reporter, and their transcriptional effects are measured by sequencing. Using this approach, we measure gene silencing and activation for thousands of domains. We find a relationship between repressor function and evolutionary age for the KRAB domains, discover that Homeodomain repressor strength is collinear with Hox genetic organization, and identify activities for several domains of unknown function. Deep mutational scanning of the CRISPRi KRAB maps the co-repressor binding surface and identifies substitutions that improve stability/silencing. By tiling 238 proteins, we find repressors as short as ten amino acids. Finally, we report new activator domains, including a divergent KRAB. These results provide a resource of 600 human proteins containing effectors and demonstrate a scalable strategy for assigning functions to protein domains.
View details for DOI 10.1016/j.cell.2020.11.024
View details for PubMedID 33326746
High-Throughput Affinity Measurements of Transcription Factor and DNA Mutations Reveal Affinity and Specificity Determinants.
Transcription factors (TFs) bind regulatory DNA to control gene expression, and mutations to either TFs or DNA can alter binding affinities to rewire regulatory networks and drive phenotypic variation. While studies have profiled energetic effects of DNA mutations extensively, we lack similar information for TF variants. Here, we present STAMMP (simultaneous transcription factor affinity measurements via microfluidic protein arrays), a high-throughput microfluidic platform enabling quantitative characterization of hundreds of TF variants simultaneously. Measured affinities for ∼210 mutants of a model yeast TF (Pho4) interacting with 9 oligonucleotides (>1,800 Kds) reveal that many combinations of mutations to poorly conserved TF residues and nucleotides flanking the core binding site alter but preserve physiological binding, providing a mechanism by which combinations of mutations in cis and trans could modulate TF binding to tune occupancies during evolution. Moreover, biochemical double-mutant cycles across the TF-DNA interface reveal molecular mechanisms driving recognition, linking sequence to function. A record of this paper's Transparent Peer Review process is included in the Supplemental Information.
View details for DOI 10.1016/j.cels.2020.11.012
View details for PubMedID 33340452